Protein Isolation
Protein isolation is an important tool in biological research, clinical diagnostics and the production of pharmaceuticals, especially production by recombinant techniques. The scientific researcher must obtain a protein quickly while retaining high specific activity; the clinician must identify proteins in biological samples in order to make an accurate diagnosis; and the molecular biologist must recover and purify large quantities of proteins produced by recombinant organisms.
Scientists have traditionally isolated proteins by precipitating them from biological samples with salts, such as ammonium sulfate, or organic solvents, such as ethanol. Exposure to the chemicals used in such methods often causes protein denaturation. In addition, separation of the proteins from the precipitating chemical is difficult and may cause further denaturation.
The ammonium sulfate precipitation technique, also known as "salting out," is based on the fact that the solubility of most proteins decreases at high electrolyte concentration. Sulfate is used because multivalent ions are more effective than monovalent ions. This procedure is usually carried out in the cold (0.degree.-4.degree. C.) with control of pH close to neutrality. Different classes of proteins precipitate depending on the concentration of salt added. The disadvantage to this method is the difficulty of removing residual salt from the precipitate or supernatant. Often dialysis is used, but is very time consuming.
Organic solvents are often used for fractional precipitation of proteins. However, there is a risk that the solvent will denature the protein unless kept at a temperature near the freezing point. In addition, the solvent must be removed from the protein. A solvent such as ethanol is generally removed by lyophilizing the precipitated proteins.
Recent advances in protein purification have centered around the development of high performance ion exchange, affinity chromatography, hydrophobic interactions and gel filtration chromatography. The biological sample is loaded onto a chromatography column and is eluted with the appropriate solvent into fractions that are analyzed for protein activity. This method is expensive, time consuming, and poorly suited for large scale protein purification.
Many scientist continue to use traditional methods alone or in combination with the recently developed chromatography procedures. For example, after precipitation of a protein from a biological sample with ammonium sulfate, protein is separated from the ammonium sulfate salt by chromatography. Often the protein loses activity or becomes denatured during one or more steps of the procedure, resulting in a low yield or inaccurate identification.